Mycobacterium tuberculosis is the causative agent for the disease tuberculosis (TB). TB kills about 2 million people each year and around a third of the world's population is infected with TB. TB newly infects nearly 1% of the world's population each year. TB is a leading cause of death among people with HIV/AIDS. Though TB can normally be treated with antibiotics, a serious problem in the worldwide fight against TB is the emergence of multi-drug resistant strains of TB. In order to develop logical targets for new drugs, the physiology of Mycobacteria must be better understood. Pathogenic bacteria such as M. tuberculosis secrete proteins in order to evade host defense mechanisms and survive in host cells, making protein export a logical drug target. The long-term goal of this work is to characterize the Sec-dependent protein export of M. tuberculosis. The Sec-dependent translocation pathway that involves dimeric SecA and SecYEG is used for export of many proteins. SecA, an essential protein found in all bacteria, is an ATPase and provides the energy used in the export of proteins through the SecYEG membrane translocation machinery. Recently, several pathogenic microorganisms, including M. tuberculosis, have been discovered to carry two SecA proteins, SecA1 and SecA2. SecA1 is essential for general protein export while SecA2 is specific for secretion of some virulence proteins. However, why these microorganisms require two SecA proteins is not understood. Because SecA proteins are generally specific to a particular microorganism, SecA1 and SecA2 provide good targets for drug development. Therefore, we propose to characterize the function of each of these SecA proteins in protein export from M. tuberculosis. We will characterize the SecA proteins using biochemical and biophysical techniques including ATPase binding and hydrolysis assays, analytical ultracentrifugation to monitor subunit associations, and develop in vitro membrane binding and translocation assays for M. tuberculosis proteins. The specific questions for this R03 grant are: 1). Do SecA1 and SecA2 possess similar abilities to bind and hydrolyze ATP?; 2) Do SecA1 and SecA2 interact with each other?; and 3.) How do SecA1 and SecA2 interact with membrane translocation machinery? These experiments are designed to initiate an in depth characterization of the function of both SecA proteins in M. tuberculosis. Mycobacterium tuberculosis is the causative agent for the disease tuberculosis (TB). TB kills about 2 million people each year and around a third of the world's population is infected with TB. TB newly infects nearly 1% of the world's population each year. TB is a leading cause of death among people with HIV/AIDS. Though TB can normally be treated with antibiotics, a serious problem in the world-wide fight against TB is the emergence of multi-drug resistant strains of TB. In order to develop logical targets for new drugs, the physiology of Mycobacteria must be better understood. [unreadable] [unreadable] [unreadable]